Biology Reference
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miR binding
sequence
Luciferase
Active small
molecule
Inactive small
molecule
miRNA
miR binding
sequence
miR binding
sequence
Luciferase
Luciferase
Luminescence
Fig. 2
Cell-based assay for small-molecule miRNA modulators. Using either a
GFP or a luciferase reporter with a miRNA-binding sequence in the 3
UTR.
Inactive molecules fail to inhibit miRNA binding and suppression of the gene,
resulting in a lack of signal from the reporter protein. Active molecules inhibit
miRNA maturation or function abrogating binding, resulting in increased levels of
reporter protein expression
′
the establishment of stable cell lines and the lack of a cellular viability
control. In order to rectify these issues, Deiters et al. modifi ed the
assay to employ a psiCHECK-2 plasmid that was transiently trans-
fected into the cell line of choice [
41
]. Moreover, the plasmid con-
tains two luciferase genes, with the miRNA-binding site only in the
3
UTR of the
Renilla
luciferase, leaving the fi refl y luciferase to act
as both a transfection and cellular viability control. To investigate
this assay system, miR-122 was selected due to its overexpression
in hepatitis C infections and under-expression in hepatocellular
carcinomas. The improved assay yielded two miR-122 inhibitors,
2
and
3
, determined by a reproducible increase in luciferase expres-
sion. Moreover, the assay also afforded a miR-122 activator (
4
) as
the control reporter system allowed for an effective measurement
of decreased levels of luciferase expression (Fig.
3
). Both the inhi-
bition and activation of the compounds were confi rmed by qRT
PCR assays, indicating alterations in levels of both the miR-122
and pri-miRNA-122. Again, the assay indicated some specifi city
for miR-122, suggesting some level of transcriptional control as
opposed to regulation of the general miRNA biogenesis pathway.
Gratifyingly, compounds
2
and
3
were able to reduce viral loads of
HCV, while
4
was able to induce apoptosis in a hepatocellular car-
cinoma cell line, suggesting the potential therapeutic values of
these miRNA-regulating compounds. However, additional
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